Plasminogen activation
Plasminogen is the precursor or inactive form of the proteolytic enzyme plasmin. Plasminogen is widely distributed in human tissues, fluids and the blood. Plasmin is the important enzyme that dissolves blood clots in the circulation. This enzyme also plays a role in tumor cell growth and spread, in implantation of the embryo and other physiological functions. Plasminogen is activated to plasmin by other proteolytic enzymes known as plasminogen activators. Two major groups of plasminogen activators have been described and partially characterized. The first is urokinase-type plasminogen activator. The prototype enzyme urokinase (UK) has been isolated from human urine or tissue culture of kidney cells. UK activates plasminogen to plasmin in purified systems and in blood, and is commercially available and used to achieve clot (thrombus) dissolution in patients. Many different tissues as well as tumors in tissue culture appear to synthesize UK or a UK-type activator.
The second type of plasminogen activator is non-urokinase enzyme. This tissue plasminogen activator is produced by some tumors in culture, notably a line of human melanoma cells, as well as by human uterine tissue. This type of activator appears also to be produced by the human blood vessel wall. This non-urokinase-plasminogen activator, to be called tissue-type or vascular plasminogen activator, activates plasminogen upon a fibrin surface, whereas the urokinase type activators do not require a fibrin clot network to activate plasminogen to plasmin. (The nomenclature "tissue type plasminogen activator" was adopted by the Subcommittee on Fibrinolysis of the International Committee on Thrombosis and Haemostasis in July 1982.)
Methods to measure plasminogen activator activity are available which are imprecise and semi-quantitative and which do not clearly distinguish between the two general types of plasminogen activators, both of which may occur in blood and in other body fluids. These methods generally include preparing a euglobulin fraction from plasma by diluting plasma at acid pH, and then testing the plasmin activity of this fraction on a fibrin or other suitable substrate. This methodology is susceptible to many variables, and provides inconsistent results.
Arterial thrombosis may be the major cause of acute myocardial infarction, Hammer, Wien Med. Wochenschr., 28: 97-102 (1878) and De Wood et al, N. Engl. J. Med., 303: 897-902 (1980) and stroke, Hunt, Am. J. Med. Sci., 147: 704-713 (1914) and Genton et al, Stroke, 8: 159-175 (1977). The thrombotic process in these states can be initiated by platelet aggregation at sites of atherosclerotic obstruction, Folts et al, Circulation, 54: 365-370 (1976) and Gertz et al, Circulation, 63: 476-486 (1981) followed by coagulation and the formation of an occlusive fibrin clot; De Wood et al., N. Engl. J. Med., 303: 897-902 (1980). Because aspirin prevents platelet aggregation by inhibiting cyclooxygenase and resultant production of thromboxane A.sub.2, Vane, Nature New Biol., 231: 232-235 (1971) and Smith et al, Nature New Biol., 231: 235-237 (1971), it has been utilized in attempts to alter the natural history of atherosclerotic, Coronary Drug Project Research Group, J. Chron. Dis., 29: 625-642 (1976), Elwood et al, Lancet., i: 436-440 (1974), Elwood et al, Lancet., i: 436-440 (1974), Elwood et al., Lancet., ii: 1313-1315 (1979), Aspirin Myocardial Infarction Study Research Group, JAMA., 243: 661-669 (1980) and Persantine-aspirin Reinfarction Study Research Group, Circulation., 62: 449-461 (1980) and thromboembolic disease; Harris et al., N. Engl. J. Med., 297: 1246-1249 (1977) and Canadian Cooperative Study Group, N. Engl. J. Med., 299: 53-59 (1978). Few of these studies individually have successfully demonstrated a significant benefit from aspirin. The Canadian Cooperative Study showed that aspirin reduced the risk of stroke and death only in male patients; Canadian Cooperative Study Group, N. Engl. J. Med., 299: 53-59 (1978). Similarly, Harris et al. showed that aspirin reduced the risk of postoperative thromboembolism, but once again only in men; Harris et al., N. Engl. J. Med., 297: 1246-1249 (1977). None of the large trials of aspirin for the secondary prevention of myocardial infarction demonstrated statistically significant benefits from this drug, Coronary Drug Project Research Group, J. Chron. Dis., 29: 625-642 (1976), Elwood et al., Lancet., i: 436-440 (1974), Elwood et al., Lancet., ii: 1313-1315 (1979), Aspirin Myocardial Infarction Study Research Group, JAMA. 243: 661-669 (1980) and Persantine-aspirin Reinfarction Study Research Group, Circulation, 62: 449-461 (1980), though beneficial trends were described.
While the effect of aspirin on platelets has been studied extensively, its effect on coagulation and fibrinolysis has been less well defined. Menon, N. Engl. J. Med., 296: 525-529 (1977) and Moroz, N. Eng. J. Med., 296: 525-529 (1977) both demonstrated an increase in whole blood fibrinolytic activity after ingestion of 1.8 g of aspirin, but Moroz showed that this was due entirely to the fibrinolytic action of cellular elements. In constrast, Ghezzo et al., Acta. Haemat., 65: 229-232 (1981) showed a decrease in fibrinolysis induced by both cellular and plasma factors after ingestion of 1 g of aspirin per day for four days. Studies in animals have shown both an antithrombotic, Housholder, et al., J. Oral. Surg., 38: 412-416 (1980) and Zimmerman et al., Artery., 8: 422-425 (1980) and a "paradoxical" thrombotic effect of aspirin; Zimmerman et al., Artery. 8: 422-425 (1980), Kelton et al., J. Clin. Invest., 62: 892-895 (1978), Zimmerman et al., Thromb. Res., 16: 843-846 (1979).
Fibrinolysis and the subsequent dissolution of blood clots depends on the formation of plasmin from the inactive precursor plasminogen by plasminogen activators; Astrup, Fed. Proc., 25: 42-51 (1966), Wallen, Progress in chemical fibrinolysis and thrombolysis. New York, Raven Press, 3: 91-98 (1978) and Collen, Thromb. Haemostas., 43: 77-89 (1980).
The method of the invention makes it possible, for example, to study the effect of aspirin or other drug which has multiple effects including an effect upon fibrinolysis induced by plasminogen activators and to select a dosage which, if possible, has desired effects while minimizing adverse fibrinolysis effects.